Imaging mass spectrometry of intraspecies metabolic exchange revealed the cannibalistic factors of Bacillus subtilis

Abstract

During bacterial cannibalism, a differentiated subpopulation harvests nutrients from their genetically identical siblings to allow continued growth in nutrient-limited conditions. Hypothesis-driven imaging mass spectrometry (IMS) was used to identify metabolites active in a Bacillus subtilis cannibalism system in which sporulating cells lyse nonsporulating siblings. Two candidate molecules with sequences matching the products of skfA and sdpC, genes for the proposed cannibalistic factors sporulation killing factor (SKF) and sporulation delaying protein (SDP), respectively, were identified and the structures of the final products elucidated. SKF is a cyclic 26-amino acid (aa) peptide that is posttranslationally modified with one disulfide and one cysteine thioether bridged to the α-position of a methionine, a posttranslational modification not previously described in biology. SDP is a 42-residue peptide with one disulfide bridge. In spot test assays on solid medium, overproduced SKF and SDP enact a cannibalistic killing effect with SDP having higher potency. However, only purified SDP affected B. subtilis cells in liquid media in fluorescence microscopy and growth assays. Specifically, SDP treatment delayed growth in a concentration-dependent manner, caused increases in cell permeability, and ultimately caused cell lysis accompanied by the production of membrane tubules and spheres. Similarly, SDP but not SKF was able to inhibit the growth of the pathogens Staphylococcus aureus and Staphylococcus epidermidis with comparable IC 50 to vancomycin. This investigation, with the identification of SKF and SDP structures, highlights the strength of IMS in investigations of metabolic exchange of microbial colonies and also demonstrates IMS as a promising approach to discover novel biologically active molecules.etabolic exchange describes the process of exchanging signals or nutrients between cells or populations and is a common feature of all living systems. Bacteria produce a wide array of signaling molecules to control metabolic as well as morphological and developmental changes in either an interspecies or intraspecies manner (1). Bacillus subtilis, for example, has a complex life cycle and thrives in diverse living conditions ranging from soil, contaminated wounds, and the intestinal tract (2-4). To accommodate this, B. subtilis dedicates ∼10% of its genome to the production of specific molecules involved in intra-and interspecies metabolic exchange (5). Two of these molecules are sporulation delaying protein (SDP) and sporulation killing factor (SKF), which, based on genetic experiments, are proposed to lyse a subpopulation of B. subtilis cells to provide nutrients for the remaining cells, a process referred to as bacterial cannibalism (6-10). This behavior is dependent on Spo0A, a master transcriptional regulator that also controls biofilm formation and sporulation (6-13).We set out to characterize these cannibalistic compounds to establish their roles in the B. subtilis life cycle and to understand their structure ...

Novel Phenol-soluble Modulin Derivatives in Community-associated Methicillin-resistant Staphylococcus aureus Identified through Imaging Mass Spectrometry

“…Additionally, the strains S. epidermidis ATCC35984 (17) and group A Streptococcus serotype M3 strain 4041-05 (Centers for Disease Control) were studied. All strains were grown in Todd Hewitt broth or Todd Hewitt agar (THA) except S. epidermidis, which was grown in tryptic soy broth.…”

Cannibalism stress response in Bacillus subtilis

“…Was it the mature toxin itself or could the unprocessed precursor also lead to its activation? SKF is a ribosomally synthesized AMP and requires post-translational modification to be fully active (González-Pastor et al, 2003;Liu et al, 2010). Our knowledge of this process is still limited and direct evidence for the functions described in the following text is still lacking.…”

“…Our knowledge of this process is still limited and direct evidence for the functions described in the following text is still lacking. However, it is assumed that the radical S-adenosyl-methionine enzyme SkfB mediates the first step in SKF maturation by forming a thioether bond between the cysteine residue Cys4 and the a-carbon of the methionine residue Met12, resulting in pre-SkfA (Flühe et al, 2013;Liu et al, 2010) (Fig. 3).…”

“…3). SkfH, a putative thioredoxin oxidoreductase-like protein and the last gene encoded in the skfA-H operon, is presumed to mediate the formation of a disulfide bond leading to SkfA * (Liu et al, 2010) (Fig. 3).…”

The predatory life cycle of Myxococcus xanthus

“…xanthus is able to lyse a wide range of Gram-negative and Gram-positive bacteria in a non-species-specific manner (Mendes-Soares & Velicer, 2013). Other soil microbes show cooperative traits, such as Bacillus subtilis, which forms specialized cell types and secretes secondary metabolites that exhibit antibiotic potential at high concentrations (Vlamakis et al, 2008;Liu et al, 2010). However, it is often unclear whether the studied organism actually produces sufficient concentrations of these metabolites for lysis in situ, nor is it obvious whether the organism then derives growth substrates directly from its lysed neighbours.…”